PROJECT SUMMARY In vivo screening of phage libraries in live mice has been used to identify peptides that direct phage homing to a tumor. The power of in vivo phage screening is illustrated by the recent discovery of peptides with unique tumor- penetrating properties. These peptides activate an endocytic transport pathway related to but distinct from macropinocytosis. They do so through a complex process that involves binding to a primary, tumor-specific receptor, a proteolytic cleavage, and binding to a second receptor. The second receptor, neuropilin-1 (or neuropilin-2) activates the transport pathway. This trans-tissue pathway, dubbed the CendR pathway, mediates the extravasation and transport through tumor tissue of payloads ranging from small molecule drugs to nanoparticles (NPs). The CendR technology provides a solution to a major problem in tumor therapy, nanotherapy in particular, which is poor penetration of drugs into tumors. Targeting with these peptides has been shown to specifically increase the accumulation of NPs in experimental tumors in vivo, and into human tumors ex vivo. Remarkably the payload does not have to be coupled to the peptide; the peptide activates a bulk transport system that sweeps along NPs present in the blood. Treatment studies in mice have shown improved anti-tumor efficacy and less damage to normal tissues with nanoparticle drugs such as Abraxane (Nab-paclitaxel) and Doxil (doxorubicin-loaded liposomes). Tumor-homing peptides, including tumor-penetrating peptides, are particularly well suited for NP targeting because the multivalent presentation of a peptide on NPs makes up for the relatively low affinity of such peptides through the avidity effect, enabling potent homing to a target. High-throughput screening has been used to identify chemical compounds that mimic the binding specificities of the tumor-penetrating peptides. We have shown that a peptidomimetic compound, despite its modest affinity, can be suitable for NP targeting without further optimization. Moreover, drugs coupled to the surface of NPs may benefit from a similar effect, as has been shown for one compound. An increase of inherent functional activities of a peptide, such as the anti-metastatic effect of the CendR peptide iRGD, can also ensue. A recent study provides insight to the nature and regulation of the CendR pathway, but important aspects of the pathway, such as the mechanism of transport from one cell to another, remain unknown. Full utilization of the CendR technology will require greater understanding of the pathway and improved compounds for targeting through this pathway. This application proposes using siRNA and in vivo imaging studies to elucidate the mechanisms of the CendR transport in tissue, as well as identification of CendR-active chemical compounds for improved targeting of NPs through this pathway. New technology for nanoparticle targeting is expected to ensue.